The envelope glycoprotein (Env) of human immunodeficiency virus (HIV-1) plays a central role in the pathogenicity of AIDS but the oligomeric valency of the protein is still not firmly established. Scanning transmission electron microscopy (STEM) has been applied to determine the subunit arrangement of purified, virion-associated Env. Mass measurements were made on virion-derived protein, obtained by large-scale virus isolation and cross-linking prior to detergent extraction. Macromolecular complexes were adsorbed onto thin carbon supports and plunge-frozen, freeze-dried and imaged in dark-field STEM at low electron dose. Images were analyzed quantitatively using a calibration standard of tobacco mosaic virus to determine the distributions of molecular weights and hence the oligomeric states of the proteins. From the mass measurements, it was determined that the complexes of HIV-1 Env were composed of trimers. This finding was further supported by the observation of triangular or tri-lobed structures in electron micrographs. The results were also confirmed by sedimentation data obtained from analytical ultracentrifugation.